BACTERIA

Classification, properties and

relevant bacteria in dentistry

Kumutha Malar Vellasamy

Department of Medical Microbiology
Faculty of Medicine
University of Malaya
50603 Kuala Lumpur
kumuthamalar@um.edu.my
 BASIC LEARNING OBJECTIVES
• General structure of bacteria
• General Phenotypic Classification of Bacteria
 Gram-positive bacteria
 Gram-negative bacteria
 Miscellaneous bacteria
 Aerobic and Anaerobic bacteria
• Pathogenesis
• Microbiological diagnosis
 Prokaryotes vs Eukaryotes
• All living organisms can be broadly divided into two
groups — prokaryotes and eukaryotes.
• distinguished by the relative complexity of their cells.
Eukaryotic cells
• animals, plants, fungi, and protists
• contain double-membrane bound nucleus with DNA
chromosomes
• contain membrane-bound organelles that compartmentalise
the cytoplasm and perform specific functions
Prokaryotic cells
• bacteria and archaea
• no nucleus or other membrane-bound organelles
 General Structure of Bacteria
Contains water,
Additional genetic Circular DNA – genetic enzymes,
Site of protein
information material ribosomes and
synthesis
circular DNA

Structure that
completely surrounds Thin rigid
the protoplam filament –
allow
- Slime layer movement
(polysaccharide)
- Allow binding to Hair-like appendage
cell surfaces - Involved in bacterial conjugation
- Avoid - Attachment of bacteria to
phagosytosis mucosal cell surface
 Classification of bacteria
• Various classification method

Cell wall composition

 Classification - Gram stain (Cell wall)
• Complex, semi rigid structure
• Enable bacteria to be classified into two major groups
 based on cell wall composition and reaction to Gram
stain
• thick peptidoglycan layer
Gram-
• retains the crystal violet
positive
• stains purple

• Thin peptidoglycan layer

• does not retain the crystal violet
Gram-
during the decoloring process
negative
• Stains pink (counter stains with
safranin)
Gram-positive vs Gram-negative cell wall

Gram-positive
Gram-negative
 Gram stain examples
Gram-positive Gram-negative

Staphylococcus Neisseria
aureus gonorrhoeae

Clostridium Escherichia coli

perfringenes
 Classification - Shape
• Bacteria vary in shape, size and arrangement
• Typically described by one of the 3 shapes described earlier
Coccus
• Spherical

Bacillus
• Rod
• Cocobacillus (short and plump)
Spirillum
• Helical
• Twisted rod
• Spirochete (spring-like)
 Aerobic vs Anaerobic bacteria
• During breakdown of oxygen, highly reactive molecules are formed (hydrogen
peroxide, superoxide free radical)
• Potential to harm/kill the bacteria
• Bacteria have acquired/possess various enzymes (catalase, peroxidase &
superoxide dismutase) to break down these toxic oxygen compounds.
 Common oral infections
1. Juvenile Periodontitis

• “Periodontitis” – inflammation of the supporting tissues of the

teeth
• Cause destruction of periodontal ligament and alveolar bone
• Juvenile periodontitis - Destruction of the supporting tissues of the
teeth that become clinically significant during adolescence or early
adulthood
• May remain undetected in young individuals, until the onset of
tooth motility, drifting, spacing of the teeth, or abscess formation
• The disease progress rapidly, compared to ordinary periodontitis
 Etiological agents
• Aggregatibacter actinomycetemcomitans (Gram
negative bacilli, anaerobes)
• Tannerella forsythia (Gram negative bacilli,
anaerobes)
• Porphyromonas gingivalis
• Eikinella corrodens
• Capnocytophaga
• Bacillus spp.
• Treponema denticola (spirochetes)
Sample for laboratory diagnosis
- Clinical specimens
Saliva
Tongue coat
Subgingival plaque
Crevicular fluid

- Gram stain and culture of

blood and chocolate agar
2. Vincent’s angina

• Pharyngeal infection
• Acute ulcerative gingivitis
• “Trench mouth”
• Ulceration
 spread and involve all gingival margin
 covered by a necrotic pseudomembrane
• Pathogen involved:
 Borrelia vincentii (spirochetes) and
 Fusobacterium (Gram negative bacilli, anaerobes)
(both are normal inhabitants of oral cavity )
• Predisposing factors: Immunosuppression,
malnutrition

• Laboratory diagnosis :
 Gram-stain and culture of purulent material
 Dark field microscopy (Borrelia vincentii)
Gastrointestinal tract
•Common disease contracted through the
GI tract
 Salmonellosis (Salmonella spp.)
 Shigellosis (Shigella spp.)
 Cholera (Vibrio cholorea)
 Ulcers (Helicobacter pylori)
 Botulism (Clostridium botulinum)
 What are pathogens? How do we
determine if a particular microbe is a
pathogen?

Culturable microorganisms
1. Koch's Postulates - Robert Heinrich Herman Koch
(proposed a series of postulates that have been applied
broadly to link specific bacterial species with particular
diseases)

Virulence genes (culture-independent, also applicable

to viruses)
2. Molecular Koch’s Postulates - genetic manipulation
Koch's Postulates
1. The suspected causative
agent must be present in all
diseased individual and
absent in all healthy
individual

2. The microorganism
should be isolated from
the diseased individual
and grown in pure
culture in vitro for
several generations

3. When pure culture

is inoculated into
susceptible animal
host, typical disease
4. The microorganism must result
must again be isolated
from lesions of
experimentally
produced disease
• Many microorganisms do not meet the
criteria of the postulates but cause
diseases
– Cannot be cultured in vitro but animal models
of infection are available
• Treponema palidum (syphilis)
• Mycobacterium leprae (leprosy)
 Molecular Koch’s Postulates
(Stanley Falkow)

1. The phenotype under investigation should be significantly

associated with pathogenic strains but not with
nonpathogenic strains
2. Specific inactivation of the gene or genes associated with the
suspected virulence trait should result in measurable
decrease in pathogenicity or virulence
3. Reversion or replacement of the mutated gene with the
wild-type gene should restore pathogenicity or virulence
The Process of Infection and Disease
Establishment of Infection
• Pathogens that are not blocked
by epithelial barriers - gain
access to underlying cell layers.
• Extracellular pathogens
(bacteria and parasites ) replicate
outside host cells
• Intracellular pathogens (viruses
and other bacteria and parasites)
must enter host cells to replicate.
• In both cases, if infection is not
controlled, the pathogen may
spread locally in the tissues or
enter the bloodstream and
spread systemically
 Definitions
• Pathogen
– Any microorganism that has the capacity to cause disease.
• Pathogenicity
– Ability of an infectious organism to cause disease
• Virulence
– Quantitative measure of the pathogenicity or degree of
pathogenicity
– i.e. quantitative measurement of the ability of an
organism to cause disease
Virulence factors
- properties (i.e., gene products) that enable a
microorganism to establish itself on or within a
host and enhance its potential to cause
disease.
- include bacterial toxins, cell surface proteins
that mediate bacterial attachment, cell surface
carbohydrates and proteins that protect a
bacterium, and hydrolytic enzymes that may
contribute to the pathogenicity of the
bacterium.
 Adherence
• Attachment of pathogen to the host at the portal of entry - initial
step in infection.
• Necessary for virulence - to avoid physical and immunological
removal of the pathogen
• Pathogen utilize specialized structures or proteins on the surface
(adhesins or adhesion molecules) to attach themselves to proteins on
the host (host receptors)
• Most microbial adhesins are lipoproteins or glycoproteins
located on the glycocalyx, capsule, pili, fimbriae or flagella
• Different adhesins target different receptors on the host
cell/tissue.
• Host receptors (fibronectin, mannose, sialic acids,
glycoproteins, etc)
• Examples of adhesion molecules and their receptors.

Streptococcus pyogens
 Invasion of host cells & tissue
• Invasion
– Entry of organisms into host cells
– Active role for organisms and passive role for host cells
• Virulent microbes are characterized by the ability to penetrate tissues
of the infected organism (invasive properties).
• Advantages of entering a human cell:
- providing the pathogen with a ready supply of nutrients.
- protecting the pathogen from complement, antibodies, and other
body defense molecules
• Invasion of the host’s epithelium is central to many infectious
processes
 Example of invasins

 collagenase and hyaluronidase

 immunoglobulin A protease
 leukocidins
 M-protein
 protein A
 Surviving Host Defenses

 Pathogen evolved strategies to avoid host immune

surveillance.
 Many evasion strategies involve altering microbial
identifiers (pathogen-associated molecular patterns
or PAMPs).
 Surviving Host Defenses
 Extracellular bacteria - resistant to killing by phagocytosis
 Avoid internalisation by absorbing normal host
components to their surfaces
• S. aureus has surface protein A that binds to the Fc
portion of IgG
 produce antiphagocytic factors - to avoid phagocytosis
• Leukocidins: toxic to phagocytic white blood cells.
Produced by Streptococcus and Staphylococcus

• Extracellular surface layer: makes it difficult for the

phagocyte to engulf them, for example- Streptococcus
pneumonia, Salmonella typhi, Neisseria meningitides, and
Cryptococcus neoformans

• Some prevent fusion of lysosome and phagosome - can

survive inside phagocytes after ingestion - Legionella,
Mycobacterium, and many Rickettsia spp.
• immunoglobulin A protease
 Secretory immunoglobulin A (IgA ) is the primary form of antibody
found on human mucosal surface
 IgA protease degrades IgA, allowing the organism to adhere to
mucous membranes
 Neiserria gonorrhoeae, Haemophilus influenzae, and
Streptococcus pneumoniae.
Example of strategies Evasion of Host Cell Recognition by Pathogens
Phenotypic
Strategy Pathogen Effector Function Consequence
Encapsulation N. meningitidis Capsule Surrounds cell wall Hides immunogenic
H. influenzae determinants
P. aeruginosa
Streptococcus spp.
C. neoformans
Ig cleavage S. pneumoniae IgA1 protease Zn-metalloproteinase; cleaves IgA Inhibits detection of
Neisseria spp. IgA1 protease Serine endopeptidase; cleaves IgA opsonized target by
H. influenzae IgA1 protease Serine endopeptidase; cleaves IgA FcαR
S. pyogenes IdeS Cysteine protease; cleaves IgG Inhibits detection of
S. pyogenes EndoS Endoglycosidase; hydrolyzes N- opsonized target by
linked glycan of IgG FcγR
S. aureus GluV8 Glutamyl endopeptidase; cleaves
IgG
Complement/ N. meningitidis fHbp Binds Factor H Inhibits activation of
Ig binding S. aureus SdrE Binds Factor H and C3b alternative
S. pyogenes M protein Binds Factor H, FHL-1, and C4BP complement
B. burgdorferi OspE Binds Factor H and FHL-1 pathway
C. albicans Gpm1 Binds Factor H and FHL-1
S. aureus Protein A Binds Fc portion of IgG Scavenges IgG;
S. aureus Sbi Binds Fc portion of IgG inhibits detection of
Streptococcus spp. Protein G Binds Fc portion of IgG opsonized target by
Streptococcus spp. FOG Binds Fc portion of IgG FcγR
 Extracellular enzymes - break down and inflict damage on
tissues or dissolve the host’s defense barriers
• collagenase and hyaluronidase
degrade collagen and hyaluronic acid - allowing the bacteria to
spread through subcutaneus tissue
 Biofilms
• Matrix embedded microbial populations, adherent to each
other and / or to surfaces or inter-surfaces.
• Consist of one or more communities of microorganisms
embedded in a glycocalyx, that are attached to a solid surface.
Steps in biofim formation
Steps in Biofilm Formation
 Benefits of Biofilms to Microbes

• Help bacteria to survive in unfavorable environmental and

nutritional conditions
• Resistance to antimicrobial agents
• Increased local concentration of nutrients
• Opportunity of genetic material exchange
• Ability to communicate between bacterial population of same
and or different species
• Produce growth factors across species boundaries
 Oral Biofilms
• Oral bacteria – capable to form biofilms on distinct surfaces
including the hard and soft tissues.
Proline-rich proteins in saliva aggregate to form salivary micelle-
like globules

Absorb to clean tooth surface – forming acquired enamel pellicle

Act as foundation for the formation of multilayered biofilm

Acquired Pellicle attracts Gram-positive cocci, facultative anaerobe

(Streptococcus mutans and Streptococcus sanguis) - pioneer

Filamentous bacteria (Fusobacterium nucleatum [obligate

anaerob] and other slender rods) adhere to the primary colonizers
 Gradually, cocci replaced by filamentous form

Vibrio and spirochetes appear as the biofilm thickens

Existence of anaerobic bacteria in aerobic environment is made

possible due to the coexistence of aerobic and anaerobic bacteria

Under normal condition, dental biofilm (plaque) on the tooth

surface is harmless

Decline in host defence mechanism – disease or

immunosuppression – allow harmless commensals to become
opportunistic pathogens

Eg disease caused by biofilm community: Dental caries, gingivitis,

periodontitis, peri-implantitis, apical periodontitis
 Toxins
Toxins produced by bacteria are generally
classified into two groups

Exotoxins Endotoxins

- extracellular toxins - lipopolysaccharides

- generated by the - part of the outer cell
bacteria and wall of Gram-neg
actively secreted bacteria
- secreted to the - body's response -
outside of the can involve severe
bacterial cell inflammation
 Exotoxins

• Protein
• Can have two variety of activities
- enzymatic activities – destroy host cell
membrane
- A-B Toxin variety (subunit A-lethal action,
subunit B-binding)
Examples of Exotoxins
Cholera toxin
(Vibrio cholerae)

Activate Adenylcyclase

Increase levels of cyclic

AMP

Alter transport of ions

across the gut lumen

efflux of ions and

water

Severe diarrhea and

water loss
Tetanus toxin - tetanospasmin - produced by Clostridium tetani

Neurotransmission The inhibitory Tetanospasmin In the absence of

is controlled by the neurotransmitters does not inhibitory
balance between (e.g., GABA, interfere with neurotransmitters,
excitatory (e.g. glycine) prevent production or excitation of the
acetylcholine) depolarization of storage of neuroaxon is
and inhibitory the postsynaptic GABA or unrestrained.
neurotransmitters. membrane and glycine, but
conduction of the rather their
electrical signal. release Muscle spasm
(presynaptic
activity)
• Shiga toxin (Shigella dysenteriae)
• Shiga-like toxins [Shiga toxin-producing E. coli (STEC)
e.g. Enterohemorrhagic E. coli (EHEC)]

- Adherence to receptors of gut cells →

Shiga-like toxin secretion → lyses od
28S rRNA → inhibition of protein
synthesis → cell death → necrosis and
inflammation of the GI mucosa →
watery-bloody diarrhoea with mucus (or
dysentery)
 Endotoxins
• Lipopolysaccharides
• Gram-negative bacteria
• Derived from cells walls and often liberated when bacteria lyse
• Exert effect by stimulating macrophages to release cytokines at
high concentrations
• Symptoms: chills, fever, weakness, shock
 Endotoxins Exotoxins
Source Integral part of cell wall of Gm – Gm + & Gm – organisms.
organisms.
Chemistry Lipopolysaccharide. Protein (polypeptide).

Mechanism of Non diffusible, released on cell Diffusible, secreted by living

release lysis. cells.
Toxicity Low toxicity. Very high toxicity.
Antigenicity Poorly antigenic. Highly antigenic.

Effect of heat Stable at temperature > Unstable to temperature > 60°C,

60°C for several hours without toxicity destroyed rabidly.
loss of toxicity.
Effect of No effect, can not be converted to Convert it into toxoid
formalin toxoid. (antigenic but non toxigenic).
Specificity Not specific action (all give fever Every toxin has specific action
and shock).

Fever Produce fever in the host through Do not produce fever in the
release of interleukin-1 from host
macrophages
 Laboratory Diagnosis of
Bacteria
• Non-culturable technique
• Cultivation
• Antibody detection
 Macroscopy
• Unaided observations
• Eg. Urine analysis - direct visual observation
urine
• note quantity, color, clarity or cloudiness
• cloudy urine: infection
• dark urine color: dehydration
• red urine color: blood in the urine, hematuria
• tea colored urine : liver disease
• orange or tea colored urine: breakdown of
muscle
Sputum:
• Salivary: when it consists mainly of saliva
• Mucous: when it is mainly mucus
• Purulent: when it appears yellow as pus
• Muco-purulent: when there are visible
yellowish particles in the mucus
• Bloody: when it contains blood.
• The presence of blood should always be
noted because it is indicative of severe
disease and could interfere with the
reading of the smear
 Culture methods
• Identification of infectious agents based on
the morphology on growth media.
• size, color
 Other methods
• Antimicrobial susceptibility - Kirby-Bauer
(disk diffusion test)

• MIC – E-test
• Serology :
- ELISA
- Agglutination test
• Molecular methods
- PCR
- Real time PCR
Thank you